Composite

Part:BBa_K3924028

Designed by: Jianfan Yang   Group: iGEM21_Tsinghua   (2021-10-01)


csgA-6xHis-TFF2

This device consists of csgA as secretion peptide, Gly/Ser linker to ensure that the function of secretion peptide and therapeutic protein do not affect each other and TFF2 as therapeutic protein.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 371
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 371
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 371
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 371
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

In order to heal the intestinal tract damage, one of notable symptoms of IBD, we adopted a special therapy expressing the therapeutic proteins controllably by E.coli Nissle 1917 (EcN) in situ. The design is based on a ternary system: sensor - secretion peptide - therapeutic proteins.

Fig.1 General design of the treatment ternary system

TFF2 is one of the candidate therapeutic proteins we screened out to treat IBD, which is the effector element in the ternary system. TFFs facilitate a significant role not only in mucosal repair but also in protecting mucous epithelia from a variety of insults in the gastrointestinal tract. The potential mechanisms to treat IBD involves anti-apoptotic properties, migration and invasion, angiogenesis, and interaction with mucins[1].

Functional Verification

Fig 2. The scheme of the proof-of-concept for therapeutic proteins.

All of these proteins are worth studying, but we only chose a few proteins as a proof of concept in our actual wet lab experiments because of the time limit and the high expense of gene synthesis.
For all candidate therapeutic proteins we did codon analysis with our own software tool.(Fig 3)

Fig 3. Codon preference confident analysis of all candidate therapeutic proteins(Compared with GenSmart).

As for TFF2, the result of codon preference is shown in Fig 4.

Fig 4. Codon preference confident analysis of TFF2.

The protein expression of the TFF2 was tested using SDS-PAGE and western blot. Because the TFF2 is under the tac promoter, we managed to induce the expression of EcN RGP-csgA-TFF2 using IPTG, acquired the centrifugation sediment for SDS-PAGE and performed western blot (GADPH as internal references).(Fig 5)

Fig 5. Western blot for csgA-TFF2 (GADPH as internal references).

Reference

[1] Aamann, L., Vestergaard, E. M., & Grønbæk, H. (2014). Trefoil factors in inflammatory bowel disease. World journal of gastroenterology, 20(12), 3223–3230.

[edit]
Categories
Parameters
None